The Protein Chemistry Laboratory (PCL) at the National Cancer Institute (NCI) - Frederick provides expertise about macromolecular interactions employing surface plasmon resonance spectroscopy, development of molecular mechanism-based macromolecular screens for small-molecule inhibitors, and advanced protein chemistry techniques. These techniques include highsensitivity (picomole to femtomole) protein identification, using methods ranging from classic Edman sequencing to those that are relatively new, such as mass spectrometry. In addition, PCL provides HPLC purification and quality control of proteins and oligonucleotides used in molecular interactions and molecular screens. PCL has pioneered the use of SPR spectroscopy to analyze macromolecular interactions and ligand[unreadable]protein interactions. SPR spectroscopy analysis provide an investigator with in-depth data related to the biophysical and biochemical behavior of a target macromolecule(s) and its interactions with specific cognate molecules. We use the BIAcore(R) instrument to acquire real-time signals that detail the molecular interactions between a tethered ligand and solution analytes. The resulting signals are rich in interaction information. The PCL's scientific staff collaborates with the Computer &Statistical Services Group (Data Management Services, Inc., NCI-Frederick) to model and extract the information contained in these signals. Protein Chemistry/Characterization. PCL's unique expertise in protein chemistry allows us to work closely with NCI investigators to optimize a particular solution to a particular problem. Using our MALDI-TOF (matrix assisted laser desorption/ionization time-of-flight) mass spectrometer, we have identified proteins using in gel digestion, identified protein cross-links, protein phosphorylation sites and other modifications. Highr esolution mass spectrometry, when needed, is done in collaboration with the Laboratory of Proteomics and Advanced Technologies (LPAT). These approaches have helped us to both identify and delineate the posttranslational modifications of proteins. These are complementary techniques to the classical Edman amino acid sequencing performed in our laboratory, which gives unambiguous and quantitative results about the Nterminal amino acid sequence of a protein. Protein, Peptide, and Oligonucleotide Analysis and Purification by High-Performance Liquid Chromatography (HPLC). A very important function of the Protein Chemistry Group is the quality control of peptides/proteins and labeled nucleic acids for the PCL's Molecular Interaction Group, its Molecular Characterization of Ligand Interactions Group, and the NCI's Screening Technology Branch (Developmental Therapeutics Program). Over the last few years PCL has expanded its use of fluorescence methods to study molecular interactions. We use steady-state fluorescence anisotropy or fluorescence intensity measurements to optimize binding conditions before performing more detailed studies using surface plasmon resonance. Within the last year we have acquired a time-resolved fluorimeter that can measure the fluorescence lifetime or rotational correlation time of a fluorescent molecule. This type of analysis can provide a complementary approach to our SPR studies. We are collaborating with Dr. Karol Gryczynski at the Center for Commercialization of Fluorescence Technologies at the University of North Texas Health Science Center to develop methods and models for measuring molecular interactions using time-resolved fluorescence approaches.